Impaired vasomotor function mediated by pertubations in neurohumoral signaling and/or impaired endothelial function is the proximate cause of vascular diseases such as hypertension, coronary artery disease, stroke, erectile dysfunction, and specific vasculopathies such as Raynaud's phenomenon (RP). The identification of novel pathways that regulate vascular tone provides new targets for treatment of vascular disorders. We have recently identified a novel mechanism that modulates vascular tone. Melanospin (Opn4) are classically found in the retinal ganglion cells where they regulate circadian rhythm and sleep. We have identified a non-visual light (opsin) receptor, Opn4, in blood vessels from a number of mammalian species, and from number of vascular beds. These receptors mediated intensity-dependent vasorelaxation to light of a specific wavelength (blue 430-460nM). Preliminary data suggest that signal transduction mechanism(s) involve soluble guanylate cyclase and phosphodiesterase 6 but not protein kinase G. Light activation leads to vascular hyperpolarization a process that involves K+ channels. The receptor is likely regulated as a classic G-protein coupled receptor in that inhibition of G-protein receptor kinase prevents stimulus-dependent desensitization and significantly decrease the light intensity needed to produce a relaxation response. Finally, in vivo, blue light is able evoke a physiologic response; significantly increasing blood flow in the mouse tail artery. We hypothesize that 1) Opn4 is an important regulator of vasodilatory function and mediates a physiologic function in vivo; 2) the signal transduction mechanism in vessels mimics visual opsins of vertebrates or simple photoreceptors of invertebrates; 3) desensitization/regulation occurs by a GRK2/arrestin mechanism; and 4) this pathway is upregulated in diseases in which NO signaling (vasodilation) is impaired and vasocontriction is enhanced such as RP. In this proposal, we plan to: 1) Further characterize the potential physiologic role of Opn 4 in vasoregulation utilizing Opn4-/- mice and newly discovered Opn4 inhibitors, opsinamides, using myography in isolated vessels and laser doppler in cranial windows as endpoints in vivo; 2) Determine signal transduction mechanism/s from receptor and G protein to channel with sharp electrode measurement of membrane potential in isolated vessels using specific inhibitors and shRNA knockdown of pathway proteins; 3) Understand receptor regulation using novel pharmacologic inhibitor of GRK2, paroxetine, vascular smooth muscle-selective GRK2-/-, and arrestin-/- mice, as well as GRK2 and arrestin shRNA adenovirus knockdown in isolated vessels. 4) Determine the role of Opn4 receptors as a target in mouse models of RP and explore mechanisms mediating photorelaxation in vivo in the cutaneous circulation of healthy humans and those with primary RP using laser flow doppler and skin microdialysis. In this way we hope to gain insight into the function and dysfunction of this novel pathway in health and disease, and determine its potential as a novel therapeutic target.